Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α‐Synuclein‐Induced Cytotoxicity in Baker′s Yeast

Abstract

We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with ahighly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human a-synuclein in live yeast cells.C yclotide MCoCP4 was selected by phenotypic screening from cells transformed with am ixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in ar atio of 1:510 4 .T his demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells.

Synthesis and Target Identification of Natural Product–Inspired Compound Collections

“…Very recently, cyclic MCoTI-I was expressed in Saccharomyces cerevisiae via protein trans-splicing based on the Npu DnaE split intein. 106 Moreover, the authors demonstrated that protein trans-splicing could be used to express engineered cyclic peptides in vivo by producing a variant of MCoTI-I that was able to inhibit cytotoxicity induced by cooverexpression of α-synuclein. 106 This development highlights the great potential of inteinbased strategies, particularly protein trans-splicing, for generating genetically encoded libraries of cyclic peptides that can be screened for novel biological activities.…”

“…The resulting engineered variant was expressed in S. cerevisiae using a split intein transsplicing approach and shown to block α-synuclein-mediated cytotoxicity in a yeast synucleopathy model. 106 Aggregation of tau, a microtubule-associated protein, is also a major feature of neurodegenerative disorders, including Alzheimer's disease. 136 A six-residue segment based on residues 306-311 of tau, Ac-VQIVYK-NH2, has the ability to form fibrils reminiscent of full-length tau, and grafting this sequence into SFTI-1 yielded a series of engineered peptides that interrupted formation of hexapeptide tau fibrils in vitro.…”

Cyclotides: Plant Defense Toxins

“…For commercial use, cyclotides need to be synthesized efficiently in abundance and so far this has been done using three approaches; namely, solid phase peptide synthesis (Clark et al 2006;Daly et al 1999b;Tam and Lu 1997;Tam and Lu 1998;Tam et al 1999b), chemoenzymatic synthesis (Thongyoo et al 2006;Thongyoo et al 2007;Thongyoo et al 2008;Thongyoo et al 2009), and biological synthesis using modified inteins (Austin et al 2009;Camarero et al 2007;Jagadish et al 2015;Kimura et al 2006). Solid phase peptide synthesis requires that the C-terminal amino acid be attached to a resin via a thioester linkage, and that the sequential amino acid additions occur such that the N-terminal amino acid is a cysteine.…”

“…The technique is reliant on the concept that the residue at the C-terminus can be utilized as a recognition site for proteasemediated ligation. The third approach utilizes a host cell to carry out intein-mediated cyclization (Austin et al 2009;Camarero et al 2007;Jagadish et al 2015;Kimura et al 2006). Briefly, the linear peptide precursor with a modified N-terminal Met residue is fused in frame with a modified intein with an α-thioester residue near its N-terminal end.…”

“…This technique has successfully been used to synthesize kB1 and MCoTI-II (Austin et al 2009;Camarero et al 2004). The same intein-based cyclization system with appropriate refinements to be in sync with the eukaryotic system, has recently been used to recombinantly express the cyclotides MCoTI-I and MCoCP4 in yeast (Jagadish et al 2015).…”